Method and system for reducing the likelihood of developing liver cancer in an individual diagnosed with non-alcoholic fatty liver disease

ABSTRACT

A method for reducing the likelihood of developing liver cancer in an individual diagnosed with non-alcoholic fatty liver disease involves providing in the gut of an individual a population of beneficial bacteria selected from the group consisting of  Lactobacillus  species, and administering fiber to the individual to maintain a therapeutically effective amount of the beneficial bacteria in the gut of the individual. In certain embodiments, monoacylglycerolacyltransferase-3 (MGAT3) synthesis is inhibited to lower triacylglycerol (TAG) production, while in others, expression of diacylglycerolacyltransferase-2 (DGAT-2) is inhibited. The beneficial bacteria are preferably modified to produce increased amounts of butyrate and may also be encapsulated in a frangible enclosure. Levels of  Roseburia  are preferably increased while the levels of  Akkermansia  spp. in the individual&#39;s gut microbiome are reduced. In other embodiments, a therapeutically effective amount of a bacterial formulation comprising  Faecalibacterium prausnitzii  is administered, or a composition comprising modified  L. reuteri  bacteria having the ability to survive conditions in the duodenum or jejunum of the individual&#39;s small intestine. Other embodiments include the administration of a bacterial formulation comprising at least one of  Coprococcus, Veillonella, Roseburia, Bifidobacterium, Faecalibacterium prausnitzii  and  Prevotella.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 16/229,252, filed Dec. 21, 2018 (now U.S. Pat. No. 10,512,661,issued Dec. 24, 2019), which is a continuation-in-part of U.S. patentapplication Ser. No. 15/392,173, filed Dec. 28, 2016 (now U.S. Pat. No.10,245,288, issued Apr. 2, 2019), which is a non-provisional of U.S.Provisional Patent Application Ser. No. 62/275,341, filed on Jan. 6,2016.

The entire disclosure of the prior applications are considered to bepart of the disclosure of the accompanying application and are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention is directed to a method for reducing thelikelihood of developing non-alcoholic steatohepatitis (NASH) in anindividual diagnosed with non-alcoholic fatty liver disease involvesproviding in the gut of an individual a population of beneficialbacteria selected from the group consisting of Lactobacillus species,and at least 6 grams per day of fiber to the individual to maintain atherapeutically effective amount of the beneficial bacteria in the gutof the individual. In certain embodiments,monoacylglycerolacyltransferase-3 (MGAT3) synthesis is inhibited tolower triacylglycerol (TAG) production, while in others, expression ofdiacylglycerolacyltransferase-2 (DGAT-2) is inhibited. The beneficialbacteria are preferably modified to produce increased amounts ofbutyrate and are also encapsulated in a frangible enclosure. Levels ofRoseburia are preferably increased while the levels of Akkermansia spp.in the individual's gut microbiome are reduced.

BACKGROUND OF THE INVENTION

The human gut is perhaps one of the most complex networks in the bodyand is colonized by trillions of microorganisms including bacteria,archaea, fungi, protists, and viruses, among which bacteria are themajor inhabitants. Hepatocellular carcinoma (HCC) is one of the mostcommon malignancies in the world. Gut microbiota has been demonstratedto play a critical role in liver inflammation, chronic fibrosis, livercirrhosis, and HCC development through the gut-liver axis. Gut microbialdysbiosis accompanies the progression of alcoholic liver disease,non-alcoholic fatty liver disease and liver cirrhosis, and promotes HCCprogression. Microbial dysbiosis contributes to cancer susceptibilityvia multiple pathways. Further studies have suggested that themicrobiota and their associated metabolites are not only closely relatedto carcinogenesis by inducing inflammation and immune dysregulation,which lead to genetic instability, but also interfere with thepharmacodynamics of anticancer agents. Chronic inflammation has beenverified as a driving cause of cancer. Inflammation promotes tumorprogression and accelerates the invasion and metastasis. The generationof inflammation-associated factors can also inactivate tumor-suppressorgenes (e.g., P53 mutation). The hepatic environment is greatlyinfluenced by the pathogens or metabolites produced by the microbiota inthe GI tract through the hepatic portal venous system. Liver exerts anessential effect on the host microbial community by filtering the bloodstream as well as metabolizing and neutralizing toxins derived fromintestinal microbes. Gut microbial dysbiosis contributes tohepatocarcinogenesis because the microbiota and microbial metabolitesare detected by liver resident immune cells and are able to modifyhepatic metabolism. NAFLD is considered to be a major risk factor forHCC.

Microbial community in H. pylori-positive individuals is characterizedby an increase in the counts of Proteobacteria, Spirochaetes, andAcidobacteria, as well as a decrease in the counts of Actinobacteria,Bacteroidetes, and Firmicutes. H. pylori generally inhabits the humanstomach. However, H. pylori from the gut can reach the liver tissuethrough the blood stream of the portal vein after surviving phagocyticelimination, or by reverse migration via the duodenum. Tight junctionsof gut epithelium get degraded due to chronic inflammation. As a result,there is an increase in intestinal permeability, as well as bacterialcounts and the levels of metabolites translocated from the gutepithelium into circulation because of the chronic inflammation.

NAFLD is the new pandemic of the twenty first century, co-existing withobesity. Fatty liver is caused by an abnormality in liver metabolismthat results in the accumulation of fat. It can be seen as a consequenceof metabolic deregulation associated with energy surplus and exceededreservoir ability of adipose tissue to store fat/energy. NAFLD isstrongly associated with obesity, insulin resistance (IR)/type 2diabetes mellitus (T2DM) and the metabolic syndrome. Obesity,particularly central obesity, is highly predictive of hepatic steatosisand disease progression, being directly proportional to the increase ofbody mass index (BMI). More than two third of patients with type-2diabetes have NAFLD.

NAFLD is also associated with increased overall mortality andparticularly increased cardiovascular mortality. It is increasingworldwide, paralleling the obesity pandemic. It has been estimated thatabout one billion individuals worldwide have NAFLD. In the Western andin the Asian world, one third of the population is affected. NAFLD ispresently the third cause of liver transplantation in the United Statesand is increasing at a rate such that it will be the first cause in thenext few years.

NAFLD is the most common liver complication of irritable bowel syndromeand also affects people with ulcerative colitis and Crohn's disease.NAFLD has become the leading cause of chronic liver diseases worldwide,causing considerable liver-related mortality and morbidity. During thepast decade, it has also become increasingly evident that NAFLD is amultisystem disease that affects many extra-hepatic organ systems,including the heart and the vascular system.

Non-alcoholic fatty liver disease is a condition ranging from benignlipid accumulation in the liver (steatosis) to steatosis combined withinflammation. The latter is referred to as non-alcoholic steatohepatitis(NASH). NASH is viewed as the hepatic component of metabolic syndrome.Estimates from the USA are that 5.7% to 17% of all adults have NASH,while 17% to 33% of Americans have NAFLD. As obesity and insulinresistance reach epidemic proportions in industrialized countries, theprevalence of both NAFLD and NASH is increasing and is thereforeconsidered to be a major health hazard. Steatosis alone is considered arelatively benign condition for the liver itself and is also areversible condition. However, the transition towards NASH represents akey step in the pathogenesis, as it sets the stage for further damage tothe liver, such as fibrosis, cirrhosis and liver cancer. While themechanisms leading to steatosis are well described, little is knownabout the actual risk factors that drive hepatic inflammation during theprogression to NASH. Consequently, therapeutic options are poor.

The number one cause of death in patients with NAFLD is cardiovasculardisease, followed by malignancies and then liver disease. Despite hugeamounts of money spent on investigating its origins and prevention,there is presently no effective treatment for NAFLD. There is no clearcurative treatment for NAFLD and thus, the management of patients isdirected to controlling of co-morbidities known to promote not onlyliver disease, but also cardiovascular disease and overall mortality. Atpresent, individuals diagnosed with NAFLD are treated by focusing ondiet and exercise, in order to lose weight. Weight loss of 5% or more ofbody weight results in a NAFLD remission rate of 75%. In addition,recommendations are to have cholesterol intake lowered to 200 mg perday, whole grains emphasized and high fructose corn syrup avoided.Higher fructose consumption, in the form of soft drinks, has beenassociated with NAFLD as it is believed to promote bacterial overgrowthand hence increases the load of endotoxin that reaches the liver. Somestudies have shown a possible beneficial effect in NAFLD for very mildalcohol consumption. Coffee has also been shown to have a protectiveeffect in terms of metabolic control and NAFLD development andprogression. The effect of lipid lowering agents in NAFLD is still notcompletely understood, though some studies have suggested a mild benefitin the use of statins. The accumulation of specific lipid intermediates,including DAG, acyl-CoA, and ceramide is thought to drive theprogression of NAFLD in humans.

Existing treatments for NAFLD demonstrate various deficiencies. Forexample, available drugs such as vitamin E, pioglitazone, andpentoxifylline have borderline efficacy, but are limited by potentialside-effects and toxicities, and do not improve liver fibrosis. Weightgain is common in patients taking thiazolidinediones, and these drugscan cause fluid retention and precipitate congestive heart failure.Rosiglitazone use is also associated with increased risk of myocardialinfarction.

A genetic link to NAFLD has been studied but has not been found. NAFLDis more frequent in East Asian Indians, followed by Hispanics, Asians,Caucasians and less frequent in African Americans. While such racialdisparities are not fully understood, it is known that African Americanshave lower fructose absorption rates than Hispanics, and fructose isconsidered an important driver of liver steatogenesis.

The gastrointestinal tract harbors an abundant and diverse microbialcommunity. It is a complex system, providing an environment or niche fora community of many different species or organisms, including diversestrains of bacteria. Hundreds of different species may form a commensalcommunity in the GI tract in a healthy person, and this complement oforganisms evolves from the time of birth to ultimately form afunctionally mature microbial population.

A healthy microbiota provides the host with multiple benefits, includingcolonization resistance to a broad spectrum of pathogens, essentialnutrient biosynthesis and absorption, and immune stimulation thatmaintains a healthy gut epithelium and an appropriately controlledsystemic immunity. In settings of ‘dysbiosis’ or disrupted symbiosis,microbiota functions can be lost or deranged, resulting in increasedsusceptibility to pathogens, altered metabolic profiles, or induction ofproinflammatory signals that can result in local or systemicinflammation or autoimmunity.

Long-chain-length hydrophobic acyl residues play a vital role in amultitude of essential biological structures and processes. Amongstother functions, they build the inner hydrophobic layers of biologicalmembranes, are converted to intracellular storage compounds, and areused to modify protein properties or function as membrane anchors.Metabolic syndrome is an ever-increasing health problem among theworld's population. It is a group of intertwined maladies that includesobesity, hypertriglyceridemia, hypertension, nonalcoholic fatty liverdisease and diabetes mellitus type II (T2D).

There is a long felt but unsolved need for an effective treatment forNAFLD and the present invention is directed to a solution for thischronic and expanding disease.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to the use of variousLactobacillus species to reduce LDL, cholesterol, and triglycerides tocause an improvement and amelioration of inflammation and steatosis. Thepresent inventors believe that particular modulation of the gutmicrobiome, including the establishment and maintenance of certainbeneficial bacteria, including Lactobacillus, Bifidobacterium, andcertain Streptococcus species, forms the basis of a treatment of NAFLD,as well as NASH, and in particular, the use of particular species thathave been modified via a CRISPR system. Nonalcoholic steatohepatitis(NASH) is a more advanced form of NAFLD where liver injury has occurred,and can lead to liver failure, portal hypertension, hepatocarcinoma andcirrhosis. Even without significant changes in BMI, glucose, or LDL2,probiotic use is believed to significantly decrease ALT, AST, totalcholesterol, HDL, and TNF-α1.

Thus, in various embodiments of the present invention, the employment ofparticular probiotics as described herein, provides a treatment forNAFLD that shows improvements in intestinal dysbiosis, leading todecreasing intestinal permeability, endotoxemia and subsequentinflammation.

The most frequent cause which leads to obesity is a dysbalance betweenenergy intake and energy expenditure. The gut microbiota contributes tohost metabolism. Gut microbiota not only influence absorption anddisposal of nutrients to the liver, but also can lead to the developmentof “metabolic endotoxemia” and activation of TLR ligands, which canstimulate liver cells to produce proinflammatory cytokines, therebyinitiating inflammation and fibrogenesis, which characterize NASH.Another possible molecular mechanism implicated in NAFLD development isthe alteration in LPS-endocannabinoid (eCB) system regulatory loops andbile acid metabolism. Thus, certain embodiments of the present inventionare directed to the modification of intestinal bacterial flora byspecific probiotics to achieve a therapeutic approach for the treatmentof NAFLD.

One strategy for NAFLD treatment encompassed by the present inventionrelates to a treatment for obesity that involves manipulation of anindividual's gut microbiota. Thus, modulation of gut microbiota byprobiotic treatment or dietary intervention provides beneficial effectswith respect to body weight, influence on glucose and fat metabolism,insulin sensitivity and reduction in chronic systemic inflammation, allof which can impact the status of NAFLD. Probiotic positive effects onhost metabolism are specifically directed to beneficial levels ofLactobacillus and/or Bifidobacterium strains. For example, employment ofSaccharomyces cerevisiae var. boulardii, Enterobacter halii orAkkermansia muciniphila are used to achieve beneficial effects forobesity and NAFLD. In certain embodiments, because obstructive sleepapnea and attendant fatigue are common in patients with NAFLD, oneaspect of the present invention relates to the use of “no-snore strips”as described herein (and in more extensive pending patent applicationsincorporated herein by this reference, e.g. U.S. Pat. No. 9,445,936)such that use of such strips can beneficially modify not only thepopulations of oral bacteria, but also snoring patterns, thus providingthose suffering from NAFLD with a way to manage such condition to permitthem to address fatigue issues and to thus sleep better, exercise more,etc.

Gut bacteria alter the way individuals store fat, how levels of glucoseare balanced in the blood, and how humans respond to hormones that makeindividuals feel hungry or full. Certain population mixes of microbesset the stage for NAFLD, obesity and diabetes. The gut community in leanpeople is diverse while obese people have a gut microbe community thatis comparatively less diverse. Lean individuals, for example, tended tohave a wider variety of Bacteroidetes, a population of varied microbesthat specialize in breaking down bulky plant starches and fibers intoshorter molecules that the body can use as a source of energy.

Probiotics have physiologic functions that contribute to the health ofgut microbiota, can affect food intake and appetite, body weight andcomposition and metabolic functions through gastrointestinal pathwaysand the modulation of the gut bacterial community. Thus, in variousembodiments of the present invention, probiotics are employed, e.g.(Enterococcus faecium, Streptococcus thermophilus L. acidophilus,Bifidobacterium longum, L. plantarum and/or B. lactis) to significantlyreduce total serum cholesterol and LDL cholesterol and to improve theLDL:HDL cholesterol ratio. In particular embodiments, a CRISPR-Cassystem (Clustered Regularly Interspaced Short PalindromicRepeats-CRISPR-associated) is employed to alter one or more of thesebacteria to modify various virulence factors associated with bacteria sothat beneficial populations of bacteria inhabit an individual's oraland/or gut microbiome.

Various embodiments of the present invention relate to a compositioncapable of increasing the level of anti-oxidized low-density-lipoprotein(OxLDL) antibodies in vivo for use in the treatment or prevention ofNASH. OxLDL is an immunogenic molecule that stimulates the induction ofanti-oxLDL antibodies. Phosphorylcholine, a component of Streptococcuspneumoniae, is a major antigen in oxLDL, which is recognized byanti-oxLDL antibodies that have protective properties. One embodimentrelates to the expression of OxLDL in bacteria via employment of aCRISPR-Cas system to insert genes for OxLDL such that such modifiedbacteria produce OxLDL to therefore stimulate the induction ofanti-oxLDL antibodies, thus providing the protective effects of suchantibodies. Using the present invention, fibrosis can be decreased orprevented by the production and administration of anti-oxLDL antibodiesto avoid inflammation of the liver and to therefore treat NASH andNAFLD. While antibodies against oxLDL are known in the art, variousembodiments of the present invention relate to a new medical use of suchantibodies, as well as to methods and systems that modify gut bacteriato enhance the production of such antibodies. In other words, variousembodiments of the invention relate to a composition comprisingantibodies against oxLDL for use in the treatment or prevention ofhepatic inflammation or more in particular the treatment or preventionof NASH, and/or the use of oxLDL antibodies for the preparation of amedicament for the treatment or prevention of hepatic inflammation andin the treatment of NASH. In certain embodiments, a method of treatmentor prevention of hepatic inflammation is provided where oxLDL antibodylevels are increased by modification of particular bacteria using aClustered Regularly Interspaced Short PalindromicRepeats-CRISPR-associated system (CRISPR-Cas) or Clustered RegularlyInterspaced Short Palindromic Repeats from Prevotella and Francisella 1(CRISPR/Cpf1) system so that the bacteria is able to produce desiredlevels of oxLDL anti-bodies.

In other embodiments, the methods and systems disclosed herein aredirected to modifying the gut microbiota of an individual to amelioratethe progression of NAFLD, including reducing liver aminotransferases,total-cholesterol, TNF-α and improving insulin resistance in individualswith NAFLD. In certain embodiments, NAFLD is thus treated by modulationof the gut microbiota. Effective treatments include employing a methodof populating a subject's gastrointestinal tract with a diverse anduseful selection of microbiota in order to alter a dysbiosis. Variousaspects and embodiments of the invention are directed to methods andcompositions for modulation of NAFLD of an individual's gut microbiomeby using bacteria that have been treated with a CRISPR-Cas orCRISPR-Cpf1 system to reverse antibiotic resistance or to renderineffective certain virulence factors in pathogenic bacterial cell, aswell as modifying gut bacteria in a manner to make them “better” invarious ways, including an ability to outcompete other undesiredbacteria. Other various embodiments of the present invention relate tothe employment of engineered autonomously distributed circuitscontaining programmable nucleases (e.g. “programmable nucleasecircuits”) that are delivered to microbial organisms in vivo to modulatethe expression of certain antibiotic resistant and virulence factors ofparticular microbial organisms. Some embodiments employ the Type IICRISPR-Cas (Clustered Regularly Interspaced Short PalindromicRepeats-CRISPR-associated) system of Streptococcus pyogenes to reverseantibiotic resistance in a wide range of microbial organisms. In certainembodiments, the CRISPR-Cas system is used to weaken resistance ofmicrobial pathogens to existing antibiotics. The use of the CRISPR-Cassystem may be viewed as a paradigm shift in combating pathogens becauseit enables autonomous and distributed neutralization of disease at thegene level. Various aspects of the present disclosure provide methodsthat comprise modifying bacterial cells to target a gene or nucleotidesequence of interest, and in particular, genes involved in the storageof fat. Such modified bacterial cells include an engineered autonomouslydistributed circuit having at least one nucleic acid encoding aprogrammable nuclease that targets a gene or nucleotide sequencedirected to fat metabolism.

While there are medications approved for treating diseases andconditions associated with NAFLD, there are currently no medicationsspecifically approved for the treatment of NAFLD itself. Treatmentprotocols have instead been focused upon the associated conditions, suchas the metabolic syndrome. Conventional treatment of NAFLD includesweight loss, restricting dietary fat, administration of medicationsemployed in the treatment of an associated condition and administrationof medications employed in the treatment of hyperlipidemia. Manymedications employed to treat conditions associated with NAFLD arehepatotoxic.

Various embodiments of the present invention are directed to a methodfor treating NAFLD in a subject in need thereof that includesadministering a composition including a therapeutically effective amountof Prevotella, and more preferably Prevotella that has been modified,e.g. by CRISPR-Cas, in a manner that reduces the effect of at least oneof the virulence factors of such bacteria. Other embodiments involve theemployment of bacteria of the Bacteroides family that have been modifiedto reduce the amount of a ligand-activated transcription factor.

Dysbiosis in a person's gut has a significant role in the pathogenesisof human NAFLD/NASH. In various embodiments of the present invention,administration of probiotics, as well as associated fiber diets tosupport such bacteria, is involved, in some embodiments employingBifidobacterium and Lactobacillus strains. Control of the bacterialflora lowers proinflammatory cytokine production (tumor necrosisfactor-α, interleukin-6, interferon-γ) via down-regulation of thenuclear factor kappa B, and decreases oxidative stress. Probiotics canreduce the urease activity of bacterial microflora, decrease fecal pHvalue and reduces amino-acid fermentation and ammonia adsorption; reduceaminotransferases, and improve the lipid status in NAFLD patients. Eachof these may be modified via CRISPR-Cas systems employed to alternativecharacteristics of an individual's microbiome.

Microbiome research in liver disease has evolved recently as an excitingnew field. Prebiotics encompass products that promote the growth ofbeneficial intestinal microbiota. Probiotics include live microbialstrains in predefined quantities. Both prebiotics and the use ofprobiotics is involved in the various embodiments of the invention asherein described. The present invention is directed in variousembodiments directed to ways to modify the microbiota to treat hepaticsteatosis, liver inflammation, fibrosis, and developing and advancedliver disease. The purposeful manipulation of the gut microbiota is doneto address various liver diseases at both early and late disease stages.

More than 90% of the adult microbiome is composed of species belongingto four bacterial phyla: Firmicutes, Bacteroidetes, Actinobacteria, andProteobacteria. Differences exist, however, with respect to differentindividuals as well as in different habitats. For example, Firmicutesare the major species in the intestine, vagina, skin, and oral cavity,while Actinobacteria and Proteobacteria are more dominant in the oralcavity, skin, and nasal cavity. The enterotype is a classification ofthe microbiome, with the gut microbiome being classified into threeenterotypes. Each enterotype includes a dominant species selected fromthe group consisting of: Bacteroides, Prevotella, and Ruminococcus, withenterotypes being unrelated to race, residential region, or diet.

The prevalence of nonalcoholic fatty liver disease (NAFLD) overall islower in Asia than in Western countries. Urban areas in India and otherparts of Asia that have adapted a ‘Western’ diet report prevalence ratesfor NAFLD and NASH of 10-24 and 3-4%, respectively, which is similar totheir prevalence rates in the West. In addition, the prevalence of NAFLDin an obese population was similarly high in Asia and Western countries.Thus, differences in NAFLD etiology and prevalence are more closelyrelated to dietary patterns than geographic differences.

It is believed that commensal microbiota protect against biliary injuryand liver fibrosis. The present inventor believes that there is asignificant association of fatty liver with H. pylori infection. Thus,various embodiments involve the modification of an individual'smicrobiome, including H. pylori in one's stomach, to combat NAFLD andNASH. Thus use of CRISPR-Cas to render H. pylori more susceptible toparticular antibiotics is one way in which such modification may beachieved.

NAFLD is a complex disease and a treatment targeting one pathologicalprocess often also causes changes in other pathways. Prebioticsrepresent a specific type of dietary fiber that when fermented, mediatemeasurable changes within the gut microbiota composition, usuallycausing an increase in the relative abundance of bacteria thought of asbeneficial, such as bifidobacteria or certain butyrate producers.Prebiotics are usually non-digestible carbohydrates, oligosaccharides orshort polysaccharides, including inulin, oligofructose, galactofructose,galacto-oligosaccharides and xylo-oligosaccharides, all leading toincreasing the relative abundance of bifidobacteria and lactobacilli.The gut of individuals with various maladies, including obesity, harborbacteria in their gut that establishes an inflammation-associatedmicrobiome, often providing a lower potential for butyrate productionand reduced bacterial diversity. Thus, one objective of the presentinvention is to alter the microbiome of such individuals to increasebacterial diversity in their gut and to increase levels of butyrateproduction. Patients with NAFLD have small intestinal bacterialovergrowth and increased intestinal permeability. Thus, altering themicrobiome of such individuals is achieved to counter the progression ofNAFLD. In certain embodiments, one objective is to increase theproportion of Ruminococcaceae in a person's microbiome and to alsoreduce the proportion of Escherichia, e.g. by modifying Escherichia viaCRISPR-Cas to make it less viable than it otherwise would be.

Probiotics can reduce liver aminotransferases, total cholesterol, tumornecrosis factor α and improve insulin resistance in patients with NAFLD.Similarly, treatment of other diseases in the gut, like inflammatorybowel disease (IBD) is implicated with respect to modification of thegut microbiome. The concept of an altered gut microbiota or dysbiosis ispossibly the most significant development in IBD and NAFLD research inthe past decade. A definitive change of the normal gut microbiota with abreakdown of host-microbial mutualism is believed to be the definingevent in IBD and NAFLD development.

In other embodiments, one objective is to increase the levels ofLactobacillus, Leuconostoc, Lactococcus, Pediococcus and Firmicutes inan individual's gut microbiome, while reducing the levels ofBacteroidetes and Akkermansia spp. In certain other embodiments, oneobjective is to increase the levels of Prevotella and Roseburia (abutyrate-producer) in a person's gut microbiome, and especially thecolon microbiome. Other embodiments focus on increasing the levels ofBacteroides in the person's gut and decreasing the levels ofEscherichia, Lachnospiraceae and Megasphaera.

Periodontal disease is a chronic infectious disease of the tissuessurrounding the teeth that result in tooth loss. Several reports haveindicated that periodontal infection is related to NAFLD. Both NAFLD andperiodontal disease are chronic inflammatory conditions that are knownas ‘silent diseases’. Therefore, both conditions need to be detectedearly and treated under collaborative medical and dental care in orderto prevent progression to NASH. The prevalence of NAFLD in the Americangeneral adult population is 10%-40% and that of NASH is approximately2%-5%. One aspect of the present invention is directed to therelationship between periodontal pathogens, e.g. composed of P.gingivalis, and the severity of NAFLD. The eradication of periodontalpathogens, such as P. gingivalis infection, is believed to have abeneficial effect upon NASH.

Certain embodiments of the present invention are directed to a methodfor treating non-alcoholic fatty liver disease by providing to anindividual in need thereof an effective amount of a compositioncomprising modified L. reuteri bacteria, preferably using CRISPR-Casand/or Cpf1 systems, to provide such bacteria in a manner so that theyhave the ability to survive the conditions in the duodenum or jejunum ofthe small intestine. Other embodiments involve a method for treatingnon-alcoholic fatty liver disease involving establishing in the gut ofan individual a population of beneficial bacteria selected from thegroup consisting of Lactobacillus, Bifidobacterium, and Streptococcusspecies and administering at least 6 grams per day of fiber to theindividual to maintain the beneficial bacteria in the gut of theindividual. Still other embodiments are directed to a method fortreating non-alcoholic fatty liver disease by increasing oxLDL antibodylevels in an individual by modifying bacteria, preferably using aCRISPR-Cas or Cpf1 system, so that the bacteria is able to producedesired levels of oxLDL. Yet other methods involve the modulation ofNAFLD of an individual's gut microbiome by using beneficial bacteria,e.g. such as one or more of bacteria from one or more of the phylas:Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria,preferably treated with a CRISPR-Cas or CRISPR-Cpf1 system to reverseantibiotic resistance or to render ineffective certain virulence factorsin pathogenic bacterial cells. In other embodiments, an individual isadministered a therapeutically effective amount of Prevotella, and morepreferably Prevotella that has been modified in a manner that reducesthe effect of at least one of the virulence factors of such bacteria.Certain embodiments are directed to a method for treating non-alcoholicfatty liver disease involving the modifying of bacteria of theBacteroides family so that they produce reduced amounts of aligand-activated transcription factor as compared to non-modifiedbacteria. In preferred embodiments, probiotics are further provided tofeed such bacteria, with the result being improvements in levels ofdensity lipoprotein, and tumor necrosis factor-α.

The growth of microbiota communities is under control of distinctsubfamilies of host genes encoding antimicrobial peptides (AMPs). Whenbacteria colonize a given human habitat, the expression of AMPs,including .alpha. and .beta. defensins and cathelicidins, is upregulatedin order to limit the spreading of bacteria. The equilibrium between theimmune system and immunoregulatory functions of bacteria appears to be adelicate balance in which the loss of a specific species can lead to anoverreaction or suppression of the innate immune system. The maintenanceof a stable, fermentative gut microbiota requires diets rich in wholeplant foods particularly high in dietary fibers and polyphenols.Individuals colonized by bacteria of the genera Faecalibacterium,Bifidobacterium, Lactobacillus, Coprococcus, and Methanobrevibacter havesignificantly less of a tendency to develop obesity-related diseaseslike type-2-diabetes and ischemic cardiovascular disorders. Thesespecies are characterized by high production of lactate, propionate andbutyrate as well as higher hydrogen production rates, which are known toinhibit biofilm formation and activity of pathogens. Thus, in variousembodiments of the present invention, these bacterial species areselected and administered to an individual in preferred ratios thatreflect those of healthy individuals so as to attain the general balanceof bacterial populations in a person's gut. Moreover, preferablybacteria are selected that are effective in inhibiting biofilm formationand in particular, those that demonstrate a high production of lactate,propionate, butyrate and hydrogen. CRISPR-Cas and/or Cpf1 may beemployed to provide such characteristics to the selected bacterialspecies in this regard.

One will appreciate that this Summary of the Invention is not intendedto be all encompassing one of skill in the art will appreciate that theentire disclosure, as well as the incorporated references, provides abasis for the scope of the present invention as it may be claimed nowand in future applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration depicting the visual difference in appearancebetween a normal liver and a liver with non-alcoholic fatty liverdisease.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Probiotic therapies can reduce liver aminotransferases,total-cholesterol, TNF-α and improve insulin resistance in NAFLDpatients. Modulation of the gut microbiota represents a new treatmentfor NAFLD. In certain embodiments, the methods and systems as describedherein are directed to inhibiting glucosphingolipid synthesis in anindividual by provision of particular microbes effective to achieve suchinhibition. In other embodiments, an engineered autonomously distributedcircuit that contains a programmable nuclease able to target a virulencefactor or an antibiotic resistance gene of the bacterial species isemployed, whether they be Gram-negative bacterial cells, Gram-positivebacterial cells, or a combination thereof. Microbial cells may includemembers of the phyla Actinobacteria, Bacteroidetes, Proteobacteria,Firmicutes, or a combination thereof. In particular embodiments, gutbacteria are modified to address the synthesis of triacylglycerol. Thereis a direct correlation between high triacylglycerol (triglyceride; TAG)levels and the severity of metabolic syndrome. Thus, controlling thesynthesis of TAG will have a great impact on overall systemic lipidmetabolism and thus metabolic syndrome progression.

The Acyl-CoA: monoacylglycerolacyltransferase (MGAT) family has threemembers (MGAT1, -2, and -3) that catalyze the first step in TAGproduction, conversion of monoacylglycerol (MAG) to diacylglycerol(DAG). TAG is then directly synthesized from DAG by a Acyl-CoA:diacylglycerolacyltransferase (DGAT). The conversion of MAG→DAG→TAG isthe major pathway for the production of TAG in the small intestine, andproduces TAG to a lesser extent in the liver.

One aspect of various embodiments of the present invention is directedtowards the therapeutic potential of inhibiting MGAT for lowering TAGsynthesis. Elevated plasma TAG has been associated with an increasedrisk of coronary and cerebrovascular ischemic events. Excessive TAGsynthesis in the intestine due to dietary fat absorption followed byincreased accumulation of TAG in the liver and adipose plays an integralrole in the progression of metabolic disorders including obesity,insulin resistance, T2D, and fatty liver disease. Limiting TAGproduction in humans provides a way to address these metabolicdisorders.

TAG is de novo synthesized in the liver and adipose tissue, whiledietary TAG is broken down and re-synthesized in the small intestine. Inthe liver, TAG is used for very low density lipoprotein (VLDL) assembly.Newly formed VLDL is secreted into the circulatory system where ittransports neutral lipids including TAG to peripheral tissues. In thesmall intestine, dietary TAG is hydrolyzed by pancreatic lipase to FAand MAG that are re-absorbed in the intestinal lumen. Enterocytes thenre-synthesize TAG and secrete it as ApoB-containing chylomicrons thatdeliver dietary fat to tissues. Most tissues including liver and adiposeuse the G3P pathway for the synthesis of TAG. In contrast, the smallintestine predominately relies on the MAG pathway.

MGAT is a major regulator of TAG homeostasis in response to diet. Inhumans, the expression of MGATs is up-regulated in the livers ofinsulin-resistant patients who have nonalcoholic fatty liver disease(NAFLD). MGAT has related acyltransferases (DGAT) and they share similarmolecular relationships, however, MGAT genes share homology with DGAT2and not DGAT1. DGAT1 is mainly expressed in adipose and small intestinetissues; DGAT2 is expressed in liver tissue. There exists a fundamentalrole for DGAT2 in TAG biosynthesis, much more so than DGAT1.

There presently are very few therapeutics existing to treat NAFLD.Various aspects of the present invention relate to the inhibition ofMGAT2 to lower TAGs and to also reduce or eliminate the progression ofNAFLD, which ultimately progresses to NASH, and later cirrhosis. Incertain embodiments, protection of the liver from developing NASHinvolves the inhibition of MGAT2 synthesis (rather than MGMAT1). Thus,various embodiments of the present invention are directed to theprotection of the liver from developing NASH by effective inhibition ofMGAT2 synthesis by employing bacteria of an individual's microbiome,especially using bacteria modified via CRISPR-Cas systems to achieveinhibition of MGAT2 synthesis.

In still other embodiments, protection of the liver from developing NASHinvolves the inhibition of MGAT3 synthesis. The MGAT3 gene, Mogat3,exists only in higher mammals and not in rodents. Thus, employment ofbacterial systems, especially engineered gut microbes that carryinhibitors of Mogat3, is one method and system to achieve the treatment,likelihood of prevention and the prevention of NAFLD. The sequence ofMGAT3 is more homologous to DGAT2 than to MGAT1 or MGAT2. Thus, MGAT3exhibits significantly higher DGAT activity than MGAT1 and MGAT2. MGAT3expression levels increase in patients with NAFLD and its levelsdecrease after gastric bypass surgery-induced weight loss. It istherefore believed that MGAT3 plays a more important role than MGAT2 inobesity related hepatic insulin resistance and NAFLD progression inhumans. In various embodiments of the present invention either theexpression of MGAT2 or MGAT3, or both, is employed to combat NAFLD, andin particular, via the employment of modified gut bacteria viaenhancement of such expression by use of the CRISPR-Cas/Cpf1 systems asdescribed herein.

While the inhibition of human intestinal DGAT enzyme blocks TAGsynthesis completely and has led to severe fat malabsorption, the use ofDGAT1 inhibitors as anti-diabetes and/or anti-obesity agents is notfavored due to gastrointestinal side effects. One aspect of the presentinvention is directed to achieving the reduction of TAG synthesiswithout these gastrointestinal side effects.

In human liver tissue, MGAT2/MGAT3 expression is correlated with theprogression of NAFLD. In the small intestine, MGAT2 inhibition resultsin changes in TAG absorption and synthesis, as well as incretinsecretion. These actions contribute to weight loss, improvement ofinsulin sensitivity and hypertriglyceridemia, and the prevention ofNAFLD progression. While the blocking of dietary TAG absorption usinginhibitors to DGAT1 exhibit unwanted gastrointestinal side effects, thetargeting of the MAG pathway as a therapeutic for metabolic syndrome isa viable option for inhibiting intestinal TAG synthesis without suchside effects. Inhibition of intestinal MGAT2 results in dynamic changesin TAG and cholesterol absorption, which leads to the changes insystemic energy balance and gut incretin release. Inhibition of theMGAT2 isozyme in the liver improves steatosis by attenuating fataccumulation and insulin resistance. In adipose, MGAT2 inhibitionreduces fat biosynthesis and improves glucose uptake. Thus, one aspectof the present invention relates to either or both MGAT3 and MGAT2inactivation in various tissues, especially by employing modified gutbacteria as described herein, to achieve the benefits of reducing bodyweight, improving insulin resistance, decreasing hyperlipidemia, andattenuating hepatic steatosis.

Acyl-CaA:diacylglycerol acyltransferase (DGAT) catalyzes the final stepin triglyceride synthesis by facilitating the linkage of sn-1,2diacylglygerol (DAG) with a long chain acyl CoA. There are two primaryisoforms of DGAT, DGAT-1 and DGAT-2. DGAT-1 is primarily expressed inthe small intestine while DGAT-2 exhibits primarily hepatic expressionwhere its expression is insulin responsive. Inhibiting expression ofDGAT-2 significantly improves hepatic steatosis. Thus, the materials andmethods of various embodiments of the present invention can be used toalter expression of DGAT-2 for the treatment of NASH and NALFD, and toreduce hepatic insulin resistance. While certain aspects of the presentinvention can involve the use of engineered nucleases to knock outDGAT-2 in a subset of liver cells, or involve the use of an engineeredtranscription factor that can be used to down-regulate DGAT-2expression, other more preferred treatment methods employ the use ofCRISPR-Cas or Cpf1 systems to inhibit DGAT-2 expression.

In still other embodiments, the present invention is directed toaddressing primary sclerosing cholangitis (PSC), a disease that involvessevere inflammation and scarring that develops in the bile ducts and isassociated with patients who suffer from IBD. Those with PSC mayultimately require liver transplantation. The cause is not known andthere is no effective medication for PSC. Primary biliary cirrhosis is achronic inflammatory intrahepatic liver disorder that slowly destroysthe small-to-medium-sized bile ducts within the liver. When these ductsare damaged, bile builds up in the liver (cholestasis) and over timedamages liver tissue. Primary sclerosing cholangitis is a similar, butaffects the part of the bile duct that is outside of the liver. In bothdiseases, inflammation leads to progressive thickening, scarring, anddestruction of the bile ducts. The buildup of bile, bile salts, andcholesterol in the liver causes damage to cell membranes in the liver,reduced production of bile salts, and fibrosis (development of scartissue). Fibrosis is both a marker of liver damage and a potentialcontributor to liver failure. Continuing damage causes scarring orcirrhosis of the liver (the liver slowly deteriorates and malfunctions),and prevents proper liver function and impaired blood circulation in theintestines. While not bound by theory, these diseases are believed to beautoimmune diseases and the present inventor believes that modulation ofa person's gut microbiota can avoid the development of suchautoimmunity, thus providing a treatment and method of preventing PBCand PSC. It is believed that bacterial antigens translocate across aleaky and possibly inflamed gut wall into the portal and biliary systemto induce an abnormal immune response and contribute to primarysclerosing cholangitis pathogenesis.

In various embodiments, the focus of modification of an individual'smicrobiome is directed to the microbiome of the small intestine, whilein others it is directed to the colon, and in still others, to both. Onestrategy in the treatment of NAFLD is to ameliorate or turn offinflammatory triggers, with some of the main targets including cytokinessuch as tumor necrosis factor (TNF)-α, chemokines, TLR4, and the NLRP3inflammasome. Gut microorganism-derived bacterial products includingendotoxin (lipopolysaccharide), peptidoglycan, and bacterial DNA cantravel up the portal vein to activate TLR4 and TLR9 on Kupffer cells andother hepatic cell types. In turn, this activation can lead to therelease of cytokines and chemokines that promote NASH. In still otherembodiments, modulation of particular bacteria by CRISPR-Cas and Cpf1systems, for example, to address bacterial pyruvate dehydrogenasecomplex component E2 (PDC-E2) homologues in particular bacteria,including but not limited to E. coli, Novosphingobium aromaticivorans,Mycobacterium and Lactobacillus species, are used to effectively treatand/or prevent these diseases. There is believed to be a common core gutmicrobial response to chronic inflammation and immune activation, suchas observed in type 2 diabetes.

In yet other embodiments of the present invention, the present inventorssubmit that bacterial expression of RNA molecules can be employed togenerate miRNA molecules that interact with the human host mRNA duringbacterial infection. Thus, such micro-RNAs derived from bacterial RNAsare used to regulate gene expression of the human host cell involved indifferent human diseases, including NAFLD. Bacterially derived microRNAsequences can significantly regulate the expression of various humangenes and thus, enhancing an individual's gut bacteria by employingCRISPR systems to regulate microRNA sequences forms various embodimentsof the present invention. In addition to NAFLD, microRNAs are believedto be involved in many human diseases, such as cancer, diabetes,rheumatoid arthritis, and others that respond to a particular bacterialenvironment, and thus, while the present description is focused onNAFLD, it will be understood that other diseases can similarly beaddressed by employment of the systems and methods as described herein.

MicroRNAs (miRNA) are small important regulators of gene expression andare currently believed to regulate approximately 70% of human genes.More than a thousand different miRNA have been characterized in thehuman genome and they all are assumed to function by a similarmechanism: The miRNAs base-pair with target messenger RNA (mRNA) andrecruit nucleases that degrade the targeted RNA from the termini and/orinhibit translation. In cancer and many other diseases, deregulation ofgene-expression is observed and in many cases miRNAs have been shown toplay an integral part or even the causative role in disease development.According to various embodiments, the present invention concerns amethod for the treatment, amelioration or prevention of a disease ormedical disorder associated with the presence or over-expression ofmicroRNA. Therefore, in certain aspects of the invention, inhibitingmiRNA activity is a strategy to treat disease, especially NAFLD.

miRNAs are a class of highly conserved non-coding regulatory factorsthat negatively regulate more than half of the protein-coding genes inmammals, are essential to most biological processes, includingproliferation, differentiation and apoptosis, and their transcription istightly controlled. In certain embodiments, a CRISPR system and/or amodified CRISPR interference system (CRISPRi) employing inactive Cas9,may be used to reversibly prevent the expression of both monocistronicmiRNAs and polycistronic miRNA clusters. Such CRISPR-based systems arereversible and thus provide advantages over more conventional knockdowntechniques. The CRISPR/CRISPRi system may be adapted to target aparticular miRNA sequence by employing a single repression vector, oftenentailing using a 20-bp sequence and thus, such a CRISPR/CRISPRi methodis useful in the generation of vectors that target multiple miRNAs andwith reduced toxicity and can silence miRNAs with no off-target effects.Using such CRISPR systems to silence miRNAs involved in the progressionof NAFLD is therefore one focus of particular embodiments of the presentinvention.

In various embodiments, particular bacterial species are targeted formodification and use to address the treatment of NAFLD. For example, L.reuteri is well-established as one of the most ubiquitous members of thenaturally-occurring gut bacteria. Host-specific strains of L. reuterihave been documented to confer broad-spectrum protection from anassortment of microbial and chemical associated disease in humans andanimals. However, traditional probiotic therapy involves administrationof bacteria with the hope that some bacteria will survive the harshgastric conditions and colonize the colon where the bacteria willreproduce and live indefinitely. Far fewer bacteria survive in theduodenum, jejunum or ileum because of factors such as acidity, immuneresponse and bile concentration. In certain embodiments, it is believedthat bacteria must be present in the duodenum or jejunum of the smallintestine for lowering cholesterol and in particular bile acid. Thus,certain aspects of the present invention are directed to themodification of particular bacteria using CRISPR-Cas and/or Cpf1 systemsto provide bacteria having the ability to survive the conditions in theduodenum or jejunum of the small intestine. Thus, in one embodiment,CRISPR systems are employed to render certain bacteria adaptive to harshacid conditions and that are otherwise considered to be beneficial to aperson in avoiding fatty liver disease. Highly bile salt hydrolaseactive bacteria provide an improved agent for reducing serumcholesterol, serum lipids, body fat, and atherogenic index and forprophylaxis and treatment of atherosclerosis, cardiovascular andcerebrovascular diseases. Modification of an individual's gut microbesto render a significant population thereof to have enhanced degrees ofBSH characteristics is one objective of various embodiments of thepresent invention.

Oral administration of probiotics has been shown to significantly reducecholesterol levels, such cholesterol-lowering effects ascribed to BSHactivity. Deconjugated bile salts are less efficiently reabsorbed thantheir conjugated counterparts, which results in the excretion of largeramounts of free bile acids in feces. Also, free bile salts are lessefficient in the solubilization and absorption of lipids in the gut.Therefore, deconjugation of bile salts is believed to lead to areduction in serum cholesterol either by increasing the demand forcholesterol for de novo synthesis of bile acids to replace those lost infeces or by reducing cholesterol solubility and thereby absorption ofcholesterol through the intestinal lumen. Microbial BSHs function in thedetoxification of bile salts and in doing so increase the intestinalsurvival and persistence of producing strains. Thus, one embodiment ofthe present invention is directed to enhancing the BSH activity by aprobiotic bacterium to maximize its prospects of survival in the hostileenvironment of the gastrointestinal tract. Increased intestinal survivalincreases the overall beneficial effects associated with strainspossessing such BSH enhanced activities. Enhanced BSH activity benefitsprobiotic bacterium that are able to survive and perform in theintestinal milieu. BSH significantly contributes to bile tolerance andsurvival and persistence of strains in the intestinal tract. Thus,certain embodiments are directed to the manipulation of bacterialstrains to enhance the BSH activity of probiotic strains (either to overexpress a native BSH or to express or over express a heterologous BSH)to improve their survivability in the intestinal tract. Extraction offecal bacteria form a person and employing the techniques as describedherein on such native populations to enhance various aspects thereof,including for example BSH activity, and then returning such modified gutbacteria to the individual, is one method that may be used to addressNAFLD in a positive manner.

This is accomplished in various embodiments by the employment ofCRISPR-Cas and Cpf1 systems to insert BSH genes in select bacteria.Certain embodiments include the administration of bile-hydrolyzingstrains (especially those modified by CRISPR-Cas and/or Cpf1 systems) tocontrol serum cholesterol. The ingestion of probiotics as describedherein is believed to be deemed preferable to statins as a way toachieve a cholesterol-lowering therapy. Manipulation of BSH activity asdescribed herein provides for more robust probiotics (whether deliveredorally or via the fecal transplantations as described herein) withimproved competitiveness and performance. Statin drugs target many ofthe underlying inflammatory pathways involved in metabolic syndrome(MetS). Thus, certain embodiments relate to the use of CRISPR-Cassystems to modify bacteria of an individual's microbiome so that theyproduce effective levels of statin drugs. The metabolic syndrome (MetS)is comprised of a cluster of closely related risk factors, includingvisceral adiposity, insulin resistance, hypertension, high triglyceride,and low high-density lipoprotein cholesterol; all of which increase therisk for the development of type 2 diabetes and cardiovascular disease.A chronic state of inflammation appears to be a central mechanismunderlying the pathophysiology of insulin resistance and MetS. Thus invarious embodiments of the present invention, use of probiotics andprebiotics in combination, as described herein, is employed to addressthe cause of NAFLD, but that is also believed to address relatedconditions, such as MetS.

In one embodiment, the bacteria employed and that are modified viaCRISPR-Cas and Cpf1 to enhance expression of BSH include Lactobacillus,Bifidobacteria, Pediococcus, Streptococcus, Enterococcus, orLeuconostoc. In another embodiment, the Lactobacillus is Lactobacillusreuteri, optionally, Lactobacillus reuteri (NCIMB 701359), Lactobacillusreuteri (NCIMB 701089), Lactobacillus reuteri (ATCC 55148),Lactobacillus reuteri (ATCC 23272), Lactobacillus reuteri (NCIMB702655), Lactobacillus reuteri (LMG 18238), Lactobacillus reuteri (CCUG32271), Lactobacillus reuteri (CCUG 32305), Lactobacillus reuteri (CCUG37470), Lactobacillus reuteri (CCUG 44001) or Lactobacillus reuteri(CCUG 44144). In another embodiment, the Lactobacillus reuteri adheresto the gastrointestinal epithelial cells, competes for adhesion, orinhibits the binding of other bacteria due to cell surface proteins.

The human gut is a rich habitat populated by numerous microorganisms,each having a CRISPR system. In certain embodiments, the CRISPR-Cassystem may be employed to render certain bacteria sensitized to certainantibiotics such that specific chemical agents can selectively choosethose bacteria more susceptible to antibiotics, see, e.g. US Pat.Publication No. 2013/0315869 to Qimron, which is incorporated in itsentirety by this reference. Another aspect of certain embodimentsincludes making synthetic CRISPR-containing RNAs that target genes ofinterest and using them with Cas enzymes.

In various embodiments, the CRISPR-Cas and or Cpf1 system is employed tocontrol the composition of the gut flora, such as by circumventingcommonly transmitted modes of antibiotic resistance and distinguishingbetween beneficial and pathogenic bacteria. For applications thatrequire the removal of more than one strain, multiple spacers thattarget shared or unique sequences may be encoded in a single CRISPRarray and/or such arrays may be combined with a complete set of casgenes to instigate removal of strains lacking functional CRISPR-Cas/Cpf1systems. Because of the sequence specificity of targeting,CRISPR-Cas/CPF1 systems may be used to distinguish strains separated byonly a few base pairs.

There are ongoing ethical concerns arising with respect to the use ofCRISPR-Cas systems—especially as it relates to modification of the humangenome. In preferred embodiments of the present invention, however, suchissues are much less prevalent for various reasons. First, becausepreferred embodiments relate to the modification of microbes—rather thanto the human genome—and especially those microbes that show tropism forhumans, the unintended consequences of employing Crispr-Cas on organismsis lessened, if not eliminated. Moreover, use of CRISPR-Cas to alsoinsert genes that have controllable elements such that the cells arekilled by triggering the expression of such genes, is another way toreduce if not eliminate concerns about an unintended release of amodified organism. These types of controls are well known to those ofskill in the art and have been long employed, for example, by thoseinvolved in creating genetically engineered organisms, such as byinserting genes so that organisms become susceptible to variousconditions, such as temperature, antibiotic exposure, etc., such thatmicrobes that may somehow escape desired conditions will not be viable.Modifying the human genome, made possible by the CRISPR technique, hasits upsides but also equally daunting downsides. Permanent deletion ofgenes from the human genome is much more controversial than deletion ormodification of bacterial genes. Thus, one desirable aspect of thepresent invention is directed to the far less controversial modificationof gut microbes resident in the human being to promote health and totrigger the desired immune responses as described herein.

In other embodiments, the use of CRISPR-Cas systems is employed toincrease butyrate production of select bacteria. For example, F.prausnitzii, one of the most abundant species in the colon, is animportant producer of butyrate, a major product of carbohydratefermentation which is implicated in providing protection againstcolorectal cancer and ulcerative colitis. CRISPR systems are used toenhance the production of butyrate by insertion of genes into select F.prausnitzii bacteria to protect against colorectal cancer and otherdiseases.

Because CRISPR-Cas/Cpf1 acts before transcription occurs, it is able tobe employed to target regulatory and other elements on the DNA ofmicrobes that make up a person's gut microbiome. In certain embodiments,CRISPR-Cas may be employed to deliver fluorescent markers to certain DNAsequences, thus permitting one to determine whether any particularsample has been treated in accordance with the present invention, thusensuring, for example, identity of various materials, compliance withsafety issues, effectiveness of gene expression or excision, etc.permitting labeling of living cells with a desired color to discernparticular attributes and states.

Other embodiments are focused on diet as it relates to the use ofprobiotics. The gut microbiota plays a critical role in transformingdietary polyphenols into absorbable biologically active species, actingon the estimated 95% of dietary polyphenols that reach the colon.Certain embodiments rely upon the ability to deliver agents via mucosaladhesive strips, such as described, for example, in U.S. Pat. No.8,701,671, which is fully incorporated herein by this reference. Thus,in various embodiments of the present invention, the engineering ofcommunal bacteria with improved properties using a CRISPR/Cas system isemployed to provide for the enhancement of health, especially as itrelates to an individual's microbiome. In certain embodiments thepresent invention is directed to delivering to microbial cells in vivo adelivery vehicle with at least one nucleic acid encoding a gene ornucleotide sequence of interest, such method employing an RNA-guidednuclease. The microbial cells may be either or both pathogenic microbialcells or non-pathogenic bacterial cells and the gene or nucleotidesequence of interest may be a virulence factor gene, a toxin gene, anantibiotic resistance gene, or a modulatory gene, and most preferablythe nucleotide sequence of interest comprises 16S ribosomal DNA (rDNA).In various embodiments, the delivery vehicle is a bacteriophage. Thus,various embodiments of the present invention include the use ofCRISPR-Cas, with the recognition that this system can be employed tobenefit human health by modifying the bacterial and other microbecommunities that humans have long been exposed to in a fashion such thatthe beneficial aspects of such microbes can be preserved, while thedisadvantageous aspects can be “cut out” of the microbe DNA—rather thanattempting to change or modify the DNA of a human.

The present invention is one way in which human health concerns can bebenefited directly by the use of a DNA deletion system without affectingthe long term and permanent deletion of human genes. It is not believedto be obvious, let alone intuitive, that human health can be benefitedby such a DNA deletion system used in a fashion that affects only gutmicrobes in a human's system.

Another aspect of the present invention includes the ability to load orimpregnate mucosal strips with any number of active agents to achieveother desirable aspects, such as administration of particular vitamins,medicinal components, and certain CRISPR-Cas modified bacteria. In someembodiments the microbes are encapsulated within encapsulationstructures selected to provide the desired degree of adhesion to themucous membranes of the throat, gut, etc., and adapted to release theactive ingredients slowly over time in situ. These encapsulationstructures may be distributed within the base material in the stripcomposition. In one embodiment, the encapsulation structures comprisemultilamellar microparticles. The multilamellar microparticles areselected to exhibit good adhesion to the mucous membranes of the throat,and are small enough to be effectively distributed in the strip. Thestrips of the present invention provide the requisite pliability andtensile strength necessary to securely adhere to a person's mucosaltissues for at least one hour, more preferably at least two hours, andpreferably a bioadhesive polymer is selected from the group consistingof polycarbophil, carbomer, one or more acrylic polymers, one or morepolyacrylic acids, copolymers of these polymers, a water soluble salt ofa co-polymer of methyl vinyl ether and maleic acid or anhydride, acombination thereof and their salts. In certain embodiments, a mucosaladhesive strip has a coated surface for resisting bioadhesion thatincludes at least one patterned polymer including coating layer having aplurality of features attached to or projected into a base surface. Thefeatures each have at least one microscale (<1 mm) dimension and have atleast one neighboring feature having a substantially different geometry.The patterned coating layer preferably provides an average roughnessfactor (R) of from 4 to 50. The coating layer resists or enhancesbioadhesion as compared to the base surface. An article having a surfacecoating with topography for controlling bioadhesion comprises a basesurface, at least one patterned polymer comprising coating layerincluding a plurality of spaced apart features attached to or projectedinto the base surface which provide at least a first feature spacingdistance. The features each have at least one microscale dimension andat least one neighboring feature having a substantially differentgeometry. The coating layer provides an average roughness factor (R) offrom 2 to 50, preferably being from 4 to 50. The coating layer resistsor enhances bioadhesion as compared to the base surface.

Still other embodiments include the use of bacteria that have beenmodified to remove or disable one or more virulence factors of theparticular bacteria. In this regard, one aspect of the present inventionis directed to the modification of certain human-specific pathogens bytargeting one or more virulence factors thereof, preferably by usingCRISPR-Cas or CRISPR-Cpf1 systems, to excise virulence factors genes, orat least portions thereof or transcriptional or translational controlstherefore, such that such pathogenic pathogens are deprived of theirundesired pathogenic characteristics. One of skill in the art canreadily assess the number and identity of human-specific pathogens, aswell as the particular virulence factors associated therewith, and canthen, employing the CRISPR systems as referenced herein, remove, renderincapable or otherwise disable the virulence facts of suchmicroorganisms such that they no long pose a pathogenic threat tohumans. Certain embodiments provide for the delivery, via the strips asdescribed herein, of one or more of the following microorganismsselected from the group comprising Lactobacillus lactis, Lactobacillushelveticus, Lactobacillus jensenii, Lactobacillus acidophilus,Lactobacillus bulgaricus, Lactobacillus amylovorus, Lactobacillusdelbrueckii, Lactobacillus casei, Lactobacillus crispatus, Lactobacillusgasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacilluspentosus, Lactobacillus rhamnosus, Lactobacillus curvatus, Lactobacillusplantarum, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillusfructivorans, Lactobacillus hilgardii, Lactobacillus fermentum,Lactobacillus reuteri, Lactobacillus viridescens, Bifidobacteriumbifidum, and Lactobacillus ingluviei. The CRISPR-Cas system ispreferably employed to excise the virulence factors of one or more ofthe following bacteria: Lactobacillus lactis, Lactobacillus helveticus,Lactobacillus jensenii, Lactobacillus acidophilus, Lactobacillusbulgaricus, Lactobacillus amylovorus, Lactobacillus delbrueckii,Lactobacillus casei, Lactobacillus crispatus, Lactobacillus gasseri,Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacilluspentosus, Lactobacillus rhamnosus, Lactobacillus curvatus, Lactobacillusplantarum, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillusfructivorans, Lactobacillus hilgardii, Lactobacillus fermen turn,Lactobacillus reuteri, Lactobacillus viridescens, Bifidobacteriumbifidum, Lactobacillus ingluviei and preferably selected from the groupcomprising the following microorganisms deposited with the GermanCollection for Microorganisms and Cell Cultures where they are numberedas DSM 25972, DSM 25987, DSM 25988, DSM 25989, DSM 25973 and have beenin accordance with the Budapest Treaty regarding InternationalRecognition of the Deposition of Microorganisms for the purpose ofpatent deposition. In a preferred embodiment of the invention, stripscontaining effective amounts of these bacteria are provided that areattached to the soft palate region of a person's mouth or on othermucosal surfaces. Other LAB that may be employed in various embodimentsinclude the following: lactobacillus slaivarius CICC 23174;Lactobacillus plantarum CGMCC 1.557, Lactobacillus rhamnosus ATCC 53103,and Lactobacillus acidophilus ATCC 4356.

Moreover, in preferred embodiments, the microbes modified are limited tothose demonstrating human tropism such that undesired and unintendedchanges to other animals and organisms are not affected and that theonly implications of such genomic alterations of human specificpathogens are restricted to such species in a manner that is not capableof affecting other than the particular human disease at issue. This caninclude, for example, modifications and/or employment of integrons,which are a two-component genetic recombination system present in thechromosome of many bacterial species. The integron incorporates mobilegenes termed gene cassettes into a reserved genetic site viasite-specific recombination, named the Integron/gene cassette system.The integron consists of three basic elements: an integrase gene, anattachment site and a promoter. These elements can be manipulated to,for example, decrease the ability of a particular bacteria in a person'sgut from being able to effectively attach to epithelial tissue; oralternatively, to coaggregate with other bacteria.

To provide necessary and sufficient written disclosure and enablement ofthe various embodiments of the present invention, the followingreferences are incorporated by reference in their entireties: U.S. Pat.No. 9,017,718 to Tan; 20140065218 to Lang et. al.; U.S. Pat. Nos.6,599,883; 8,383,201; 5,158,789; 20070218114 to Sorousch; 20040136923 toDavidson; U.S. Pat. No. 8,999,372 to Davidson; 20090196907 to Bunick;20090196908 to Lee; 20030124178 to Haley; 20070293587 to Haley;20100285098 to Haley; 2006-0204591 to Burrell; U.S. Pat. No. 7,087,249to Burrelll; U.S. Pat. No. 6,210,699 to Acharya; U.S. Pat. No. 8,865,211to Tzannis; 20140199266 to Park; U.S. Pat. No. 6,599,883 to Romeo;PCT/US2008/080362 to Dussia; 2007-0218114 to Duggan; 20040136923 toDavidson; 20110142942 to Schobel; 20040120991 to Gardner et al.; Fuchset al. U.S. Pat. No. 4,136,162; 20040136923 to Davidson; U.S. Pat. No.4,163,777 to Mitra; U.S. Pat. No. 5,002,970 to Eby, III; 20040096569 toBarkalow et al.; 20060035008 to Virgallito et al.; 20030031737 toRosenbloom; U.S. Pat. No. 6,919,373 to Lam et al.; 20050196358 toGeorglades et al.; U.S. Pat. No. 3,832,460 to Kosti; 2002002057 toBattey et al.; 20040228804 to Jones, et al.; U.S. Pat. No. 6,054,143 toJones; U.S. Pat. No. 5,719,196 to Uhari; 20150150792 to Klingman;20140333003 to Allen; 20140271867 to Myers; 20140356460 to Lutin;20150038594 to Borges; U.S. Pat. No. 6,139,861 to Friedman; 20150216917to Jones; 20150361436 to Hitchcock; 20150353901 to Liu; U.S. Pat. No.9,131,884 to Holmes; 20150064138 to Lu; 20150093473 to Barrangou;20120027786 to Gupta; 20150166641 to Goodman; 20150352023 to Berg;20150064138 to Lu; 20150329875 to Gregory; 20150329555 to Liras;20140199281 to Henn; US20050100559 (proctor and Gamble); 20120142548 toCorsi et al.; U.S. Pat. Nos. 6,287,610, 6,569,474, US20020009520,US20030206995, US20070054008; and U.S. Pat. No. 8,349,313 to Smith; andU.S. Pat. No. 9,011,834 to McKenzie; 20080267933 to Ohlson et. al.;20120058094 to Blasser et. al.; U.S. Pat. No. 8,716,327 to Zhao;20110217368 to Prakash et. al.; 20140044734 to Sverdlov et al.;20140349405 to Sontheimer; 20140377278 to Elinav; 20140045744 to Gordon;20130259834 to Klaenhammer; 20130157876 to Lynch; 20120276143 toO'Mahony; 20150064138 to Lu; 20090205083 to Gupta et al.; 20150132263 toLiu; and 20140068797 to Doudna; 20140255351 to Berstad et al.;20150086581 to Li; PCT/US2014/036849 and WO 2013026000 to Bryan.

Another aspect of certain embodiments of the present invention isdirected to a thin film mucosal layered strip wherein modified bacteria(e.g. via the CRISPR-Cas system) is encapsulated in a frangibleenclosure and is present in an amount of at least about 0.5 ml. Othertreatment agents may be encapsulated in such strips, such thatantibiotics or co aggregation agents or LAB, etc. can be encapsulated ina manner that they can be released at a time when the person so desiresand/or when the strip dissolves to a certain extent, e.g. when the wallsof the encapsulating shell is worn thin enough to fracture to releasethe agent(s). The manner in which a capsule can be fractured in order torelease its solvent contents is variable and will be understood by thoseof skill in the art. Preferably, the capsule is constructed in a mannerthat it is sufficiently robust such that mere transport and packaging ofthe strips containing such capsules does not cause any leakage orbreakage of such capsules. Instead, the design of capsules is such thatthey are frangible with a considerable amount of force being directlyapplied thereto once the strips are placed on a particular mucosalsurface, such as on the soft palette of a human, such that the person'stongue, when pressing against such capsule, can cause it to fracture torelease the contents of the capsule. In other embodiments, two or moredifferent materials may be released.

Short-chain fatty acid production by commensal bacteria is important inregulating the immune system in the gut. Butyrate plays a direct role ininducing the differentiation of regulatory T cells and suppressingimmune responses associated with inflammation. Butyrate is normallyproduced by microbial fermentation of dietary fiber and plays a centralrole in maintaining colonic epithelial cell homeostasis and barrierfunction. Various embodiments described herein promote the production ofbutyrate via modified microbes administered to an individual, alone orin concert with the various other agents as described herein.

Preferably, the modified bacteria employed in certain embodiments of thepresent invention are administered orally to a patient in order todeliver the therapeutic directly to the site of inflammation in the gut.The advantage of this approach is that it avoids systemic administrationof immunosuppressive drugs and delivers the therapeutic directly to thegastrointestinal tract. The viability and stability of such modifiedbacteria is preferably enhanced to support the production of suchmicrobes of desired agents, e.g. tomatidine, p53 protein, etc. and bydoing so, a method is provided that reduce gut inflammation, enhance gutbarrier function, and/or treat autoimmune disorders. Preferably, suchmodified bacteria are capable of producing therapeutic anti-inflammationand/or gut barrier enhancer molecules, particularly in the presence ofreactive nitrogen species, and more preferably the bacteria arefunctionally silent until they reach an environment containing localRNS, wherein expression of the therapeutic molecule is induced. Incertain embodiments, the genetically or CRISPR engineered bacteria arenon-pathogenic and may be introduced into the gut in order to reduce gutinflammation and/or enhance gut barrier function. For example, in someembodiments, the bacteria are under the control of a RNS-responsiveregulatory region and a corresponding RNS-sensing transcription factorsuch that a desired product, e.g. butyrate, is produced, which inducesthe differentiation of regulatory T cells in the gut and/or promotes thebarrier function of colonic epithelial cells. Use of such modifiedbacteria, especially those modified via CRISPR-Cas systems, provides away to generate a desired therapeutic effect in a manner that lowers thesafety issues associated with systemic exposure.

Various embodiments of the present invention are directed to the fieldof oncology, and in particular, embodiments directed to a method ofameliorating, treating, or preventing a malignancy in a human subjectwherein the steps of the method assist or boost the immune system ineradicating cancerous cells. In certain embodiments, the administrationof beneficial bacteria to an individual's microbiome is achieved, withsuch bacteria being modified so as to produce effective amounts ofdesired compositions, compounds, agents, etc., e.g. tomatidine, p53protein, etc., to address cancerous conditions. In several embodiments,the administration of such beneficial bacteria and microbes to anindividual's microbiome invokes either an active (or a passive) immuneresponse to destroy, weaken or render less invasive certain cancerouscells. Various other embodiments are drawn to the co-administration ofbiological adjuvants (e.g., interleukins, cytokines, BacillusComette-Guerin, monophosphoryl lipid A, etc.) in combination withconventional therapies for treating cancer. In particular, theco-administration of various pre-biotic compositions to enhance andsustain the desired effects of the beneficial modified bacteria formsanother aspect of the present invention. In this regard, incorporationby reference of U.S. Patent Publication No. 2016/0213702 to Maltzahn etal. is included as part of the written description of various aspects ofthe present invention. For example, in view of the fact that themicrobiota of humans is complex and varies by individual depending ongenetics, age, sex, stress, nutrition and diet, modifying the numbersand species of gut, oral, vaginal and skin microbiota can altercommunity function and interaction with the host. A number of probioticbacteria known in the art, as well as some foods considered to be‘prebiotic’ that contain substances that promote the growth of certainbacteria and that stimulate beneficial microbiota shifts to improvehuman health, can be employed in concert with the modified bacteria asdescribed herein to effect desired cancer treatment regimens. Forexample, the administration of glycans in an amount effective tomodulate the abundance of the bacterial taxa can be used to achievebetter outcomes for cancer patients.

One application of the present invention is to provide a CRISPR-Casmodified bacteria, such as a lactobacteria or BCG, to a person diagnosedwith cancer, so as to facilitate the production of tomatidine in amanner that is effective to preserve muscle mass and function in suchindividual. Other embodiments include CRISPR-Cas, CasX, CasY, etc.modified bacteria that express levels of tumor suppressor factors, suchas p53, in a manner that provides an effective, therapeutic amount to anindividual via the production of such factors by one or more of theindividual's microbiome (e.g. gut, oral, skin, vaginal, etc.) By havingthe individual's microbiome responsible for administration of suchfactors, instead of attempting to administer such factors via moretraditional routes, such as injection, pills, etc., it is believed thata better result can be attained in a much more natural fashion.Moreover, in view of the ability to further modify bacteria in variousways to provide desired factors at particular times, or in conjunctionwith particular agents, it is possible to fine tune the administrationof desired factors, such as p53, butyrate, etc. so as to reduce anyunder or over production thereof. For example, rendering particularmodified bacteria sensitive to a predetermined antibiotic can thusprovide a way to reduce the numbers of any given modified bacteria in amanner to control the populations of such bacteria in an individual'smicrobiome, and hence, control the level of production of factorsproduced by such bacteria. To comply with written description andenablement requirements, incorporated herein by the following referencesare the following patent publications: U.S. Patent Publication Nos.2014/0349405 to Sontheimer; 2014/0377278 to Elinav; 2014/0045744 toGordon; 2013/0259834 to Klaenhammer; 2013/0157876 to Lynch; 2012/0276143to O'Mahony; 2015/0064138 to Lu; 2009/0205083 to Gupta et al.;2015/0132263 to Liu; and 2014/0068797 to Doudna; U.S. Pat. No. 8,945,839to Zhang; 2014/0255351 to Berstad et al.; 2015/0086581 to Li;PCT/US2014/036849 and WO 2013026000 to Bryan; 2016/0199424 to Berry etal.; 2013/0326645 to Cost et al.; 2018/0312851 to Falb et al.,2018/0296582 to von Maltzahn et al.; 2018/0207165 to Harmsen et al.,2018/0000878 to Goodman et al. and 2018/0326008 to Schreiber et al.;Ser. No. 16/142,171 to Kovarik and Ser. No. 15/395,419 to Kovarik

CRISPR-based genetic editing tools offer an efficient way to manipulateexpression levels of multiple genes and to provide a solution towardsthe “multivariate modular metabolic engineering”, to optimize the drugsynthesis pathways with modular, multiplex regulation using only a fewcore proteins (e.g., dCas9) that are guided to specific sequences byguide RNAs.

In still other embodiments of the present invention, modifying bacteriaso as to administer them to a person's microbiome is performed in amanner so that particular agents, factors or proteins derived from fungiand mushrooms, are rendered possible, with desired mushroom derivedcomponents believed to have anti-cancer characteristics, either alone orwhen used in conjunction with other agents. In particular, combining thereferenced ability to have bacteria within a person produce desiredamounts of tomatidine as well as having the same bacteria (or in otherembodiments, another bacteria) produce a separate cancer-fighting agent,is one novel aspect of the present invention. In particular, byassessing initially the particular bacterial constituents of anindividual's microbiome and then administering to such individual asimilar species of microbe, but one which has been modified, preferablyvia employment of a CRISPR-Cas system, one is able to effectivelyadminister to such individual various desired anti-cancer treatments ina way that is believed to be far less disruptive, efficient anddependable as compared to other routes of administration. Themodification of specially designed bacteria that reside in a person'sbody is believed to alleviate the concerns regarding genetic alterationof the human genome, as what is being modified is a microbiome that ispresent in a person's body—but is not directly involved in the humangenome itself. There are a myriad of ways to combine various triggeringfactors to turn on or off particular productions of agents, factors orproteins that may be included in such modified microbiome species. Thepresent invention in various embodiments is directed to at least thoseembodiments where cancer therapeutic agents can be administered by themicrobiome of the individual that has cancer so as to effectively treatthe cancer and/or remedy the symptoms resulting from the disease.

One aspect of the present invention is directed to the employment andmodification of an individual's microbiome to address muscle massretention and as a corollary thereof, to address the counterpart ofobesity by lessening the amount of fat storage by such individual. Incertain embodiments, the provision of effective amounts of tomatidine isrendered available to an individual via the inoculation of theindividual's microbiome (e.g. oral or gut) by particular bacteria thathave been modified to express amounts of tomatidine. Still otherembodiments also involve the reduction in the amount of acetate levelsin an individual's body, which in turn lowers the amount of insulin theindividual will produce, which has the effect of keeping fat cells fromstoring more energy in the form of fat. The reductions in the amount ofacetate available in an individual's body further reduces the amount ofthe hormone ghrelin, thus reducing the hunger drive of the individual.Thus, the modification of an individual's microbiome influences variousaspects of their metabolism in a manner that not only retains andmaintains the ability to nurture muscle tissue, but to also reduceobesity by affecting the amount of fat that the body stores. While notbound by theory, it is believed that the gut bacteria of an individualis a substantial source of acetate production. The production of acetateby gut microbes is believed to send signals to the brain of theindividual to initiate the production of insulin, conveyed via the vagusnerve. Fine tuning of the amount and type of gut microbes (e.g. via theuse of antibiotics to initially reduce the kind and numbers of undesiredbacteria, followed by purposeful inoculation of an individual's gutmicrobiome with modified microbes, e.g. via CRISPR-Cas insertion ofparticular factors, proteins, etc., such as tomatidine) is an effectiveway to address not only muscle wasting issues, but also obesity issuesof individuals.

While there are many gut bacteria that produce acetate and butyrate,particular bacteria are preferably selected and even more preferably aremodified using CRISPR-Cas systems to address the levels of acetateand/or butyrate production once such bacteria are introduced (orenhanced) to an individuals' microbiome. Preferably the gut microbiotaare members of two bacterial divisions: the Bacteroidetes and theFirmicutes, and most preferably include F. prausntizii. The modificationof an individual's gut microbiome is directed in a manner such that thetypical increase seen in the relative abundance of the Firmicutes and acorresponding division-wide decrease in the relative abundance of theBacteroidetes in obese individuals, is addressed. Obese people have moreFirmicutes and almost 90% less Bacteroidetes than the lean people.Preferably, the administration of modified Bacteroidetes is achieved tomore substantially reflect gut populations in more lean individuals, andby doing so, reducing the amount of acetate produced by the overall gutmicrobiome. Such a shift in the population of gut microbes to favorBacteroidetes over Firmicutes, whether or not coupled with theadministration of tomatidine, is one aspect of the present invention'sobjective of achieving a greater proportion of muscle mass than fat thatwould otherwise occur in any given individual. In still otherembodiments, addressing the acetate production by especially Firmicutes,which has an increased capacity for fermenting polysaccharides relativeto the lean-associated microbiome, is another way to achieve thisobjective, and addresses the significant obesity issues especiallyprevalent in Western societies.

In yet another embodiment, encapsulated structures, preferablymicroencapsulated structures, are employed that are filled with desiredagents, including but not limited to tomatidine, butyrate, etc. and/ormicrobes, especially bacteria that are found in an individual's gutmicrobiome, such as F. prausntizii, such that effective amounts of theagents can be administered to treat particular diseases. Other agentsmay include those effective in combatting cancer, such as but notlimited to tomatidine, p53 protein, statins, PTEN, rapamycin, and otheragents able to treat cancer symptoms. Preferably, the bacteria comprisebacteria that are found in the communities of healthy humans, including,for example, F. prausntizii, Streptococcus, Actinomyces, Veillonella,Fusobacterium, Porphromonas, Prevotella, Treponema, Neisseria,Haemophilus, Eubacteria, Lactobacterium, Capnocytophaga, Eikenella,Leptotrichia, Peptostreptococcus, Staphylococcus, and Propionibacterium.Such encapsulated structures may be provided as strips that may bemanufactured to have desired dissolvable aspects thereto and thatfurther have encapsulated portions that house the desired agents.

Similarly, it is desired to increase the presence in samples provided tourban dwelling expectant mothers of other bacteria, and in particular,Bacteroides-Prevotella, bifidobacteria, Desulfovibrio spp., Clostridiumclostridiforme, and Faecalibacterium prausntizii. Avoidance ofantibiotics by the expectant mother during the period to which she isexposed to the various Amish soil constituents is desired if notcritical in certain embodiments due to the profound changes due to suchduring antibiotic treatment. In other embodiments, the use of CRISPR-Cassystems is employed to increase butyrate production of these bacteria.For example, F. prausntizii, one of the most abundant species in thecolon, is an important producer of butyrate, a major product ofcarbohydrate fermentation which is implicated in providing protectionagainst colorectal cancer and ulcerative colitis.

An individual's microbiome includes the collective genomes of all themicroorganisms that are part of the body's ecosystem. As stated herein,various autoimmune diseases are capable of being ameliorated by thepractice of the present invention, including Crohn's disease. Priorresearchers have found that several specific microbes were moreprevalent in patients with Crohn's than in their healthy counterparts,while other bugs were less common in Crohn's cases. Addressing thisdysbiosis, or imbalance, in the microbial ecosystem is one aspect of thepresent invention. In certain embodiments, certain microbiota that wereknown to disappear in the guts of Crohn's cases, are reintroduced,including Faecalibacterium prausnitzii, and at the same time, severalbacteria that are known to proliferate in Crohn's cases, including thoselinked to IBD and colorectal cancer, are targeted to remove pathogenicabilities. In particular embodiments, providing a collection ofmicrobes, preferably including, for example a higher than normal (e.g.that is found in any random sampling of Amish soil) amount of Faecali,more preferably Enterococcusfaecalis, is achieved to expose expectantmothers and infants thereto in order to trigger desired immune systemresponses. Enhancing the growth and viability of this particularbacterium in the gut—and then use of such modified bacterium to treatindividuals with various diseases, such as Crohn's disease and otherautoimmune diseases. Similarly, Faecalibacterium prausnitzii, whichrepresent more than 5% of the bacteria in the intestine, is encouragedto populate the guts of patients. Such enhanced growth of this bacteriummay also be employed to combat certain forms of inflammatory boweldisease. In various embodiments of the present invention, Enterococcusfaecalis is are subjected to CRISPR-Cas procedures to remove undesiredvirulence and pathogenicity factors, such as several genes isolated fromresistant enterococci (agg, gelE, ace, cyl LLS, esp, cpd, fsrB) whichencode virulence factors such as the production of gelatinase andhemolysin, adherence to caco-2 and hep-2 cells, and capacity for biofilmformation. Deletion and removal of certain antibiotic resistance, forexample the acquisition of vancomycin resistance by enterococci, isdesired also so as to properly and safely employ this bacteria in thepresent invention. In a particular embodiment, the addition of E.faecalis LAB3 1 is employed to trigger desired immune system responses.

In certain embodiments, it may be advantageous to genetically modify agut mucosal-associated bacteria with polynucleotides and as taughtherein to express or overexpress the polynucleotides as taught herein orto produce or overproduce the polypeptides, such as butyrate andacetate, directly into the vicinity of, or within the gut mucosalbarrier of a human. In a preferred embodiment, the gutmucosal-associated bacteria may by any bacteria from the species F.prausinitzii, Prevotella intermedia, and/or Akkermansia muciniphilla.Such overproduction may be realized by genetic modification toolsinvolving recombinant DNA technologies, genome editing such as by usingtools based on CRISPR/cas-like systems, or by classical mutationselection systems.

In an embodiment, the genetically modified host cell may be anybacteria, particularly one which is not from a species of bacteria thatnaturally occurs or lives in the vicinity of or within the gut mucosalbarrier of a mammal. Non-limiting examples of such bacteria include anybeneficial isolated intestinal bacterial strains, e.g. probioticbacteria, particularly strains selected from the genera Lactococcus,Lactobacillus, or Bifidobacterium may be used. In addition, strictanaerobic intestinal bacteria may be used such as those belonging to thegenera known to occur in the human intestinal tract. As describedherein, in various embodiments, strictly anaerobic bacteria areencapsulated or microencapsulated to avoid contact with oxygen, and aredelivered to a human such that the encapsulation is dissolved orfractured to release such bacteria in a portion of the body, e.g. gut,where it can thrive.

Certain embodiments employ the bacterium Flavobacterium akiainvivens,which was discovered in 2012 on the plant Wikstroemia oahuensis, or“akia,” which is a flowering shrub endemic to Hawaii. That bacterium hasbeen found on that plant and no other. The bacterium forms 2- to3-millimeter diameter colonies that range from cream to off-white incolor and wet to mucoid in viscosity, and (it) was isolated fromdecaying Wikstroemia oahuensis collected on the island of Oahu.

Certain embodiments are directed to the targeted manipulation of the gutmicrobiome for therapeutic applications, such as the manipulation of thegut microbiome achieved by altering the microbiota population andcomposition, or by modifying the functional metabolic activity of themicrobiome to promote health and restore the microbiome balance. Therehas been recent progress in the engineering of gut commensals, whichalso presents great potential for bio-medical applications.Specifically, in Bacteroides thetaiotaomicron, components for tunablegene expression were developed and characterized and expected functionaloutputs were observed in mice after administration of these engineeredB. thetaiotaomicron. Thus, one aspect of various embodiments is toharness such engineered commensals, especially F. prausntizii for theoverproduction of butyrate, for therapeutic purposes.

F. prausntizii was first isolated in 1922 by C. Prausnitz.Morphologically, F. prausntizii is a Gram-negative, non-motile andnon-sporeforming rod with a diameter of 0.5 to 0.9×2.4 to 14.0 μm. F.prausntizii is a strictly anaerobic bacterium that produces butyrate,formate, D-lactate and CO2 but no hydrogen as fermentation products andF. prausntizii growth is inhibited by acidic pH and bile salts. Theamount of F. prausntizii in the healthy human gut is linked to diet.Inulin-derived prebiotics have been shown to significantly increase F.prausntizii concentration in the gut. F. prausntizii is statisticallylinked to eight urinary metabolites: dimethylamine, taurine, lactate,glycine, 2-hydroxyisobutyrate, glycolate, 3,5-hydroxylbenzoate and3-aminoisobutyrate. It is believed that F. prausntizii has pronouncedanti-inflammatory effects. While not bound by theory, F. prausntizii mayinduce an increased secretion of an anti-inflammatory cytokineinterleukin 10, and a decreased secretion of pro-inflammatory cytokineslike interleukin 12 and tumor necrosis factor-α production. It isfurther believed that F. prausntizii has the ability to suppressinflammation, and it is hypothesized that this is due to metabolite(s)secreted by F. prausntizii, including but not limited to butyrate. Thenumber of F. prausntizii is significantly higher in the gut of healthysubjects as compared to IBD and it is believed that F. prausntizii iscrucial to gut homeostasis and disease protection.

With the guidance provided herein, as well as the numerous referencesincorporated by reference herein, one of skill in the art willunderstand the feasibility of using engineered bacteria to directlymanipulate the functional output of the microbiota without majormodulation of the microbiota population and composition. Components inthe normal diet and/or employing prebiotics and engineered probioticsare therefore harnessed to render a targeted effect on the host throughmodulating the functional output of the microbiome.

F. prausntizii is a multi-skilled commensal organism and a chief memberof human microbiota. It is broadly distributed in the digestive tract ofmammals and also in some insects. It is rich in the hind gut rather thanin the stomach, as well as jejunum. The consumption of a higher quantityof animal meat, animal fat, sugar, processed foods, and low fiber diet(the typical westernized diet) reduces the count of F. prausntizii,while a high-fiber (vegetables and fruits) and low meat diet enhance thecount of F. prausntizii. It is known to consume a variety of dietcontaining polysaccharides, such as the prebiotic inulin, arabinoxylans,apple pectin, oligofructose, resistant starch, fructan supplement,pectins and some host-derived carbon sources (including d-glucosamineand N-Acetyl-d-glucosamine). Meta-analyses also show that the increasedconsumption of fiber significantly reduces the risk of mortality.

The discovery of the clustered regularly interspaced short palindromicrepeats (CRISPR) and the CRISPR-associated nuclease 9 (Cas9) system, hasled to an array of strategies to manipulate the gut microbiome withprecision. Engineered phage (with the CRISPR-Cas9 system) can beemployed to target pathogenic bacteria, or remove a population ofbacteria that aids pathogenic bacterial growth, thereby fine-tuning andrestoring the balance of the gut microbiome. CRISPR/Cas9 can also beused to manipulate and differentiate genetically heterogeneous bacteria,even of the same species. Unlike conventional drugs, the CRISPR/Cas9system targets specific bacteria at the gene level to selectively removepathogens, virulence factors, genes of undesired expressed proteins,etc. and can further be used as an antimicrobial adjuvant to improveantibiotic treatment. Citorik et. al. demonstrated how CRISPR/Cas9 canbe delivered using bacteriophages, targeting the ndm-1 gene, which codesfor the broad-spectrum carbapenemase, New-Delhi metallo-β-lactamase.Ndm-1 targeting CRISPR/Cas9 specifically eliminated E. coli harboringthe gene without affecting wild-type, or other, E. coli strains presentin a synthetic consortium of microbes. Other examples include there-sensitization of bacteria to antibiotics and immunization of bacteriato incoming plasmids conferring antibiotic resistance using temperatephages. Yosef et al. used CRISPR/Cas9 to target ndm-1 and ctx-M-15,which expresses a broad-spectrum beta-lactamase, and effectivelyselected the transduced bacteria that were antibiotic-sensitive. Thus,CRISPR/Cas9 may be employed to manipulate the gut microbiome bydiscriminating at the gene level to change the characteristics andfunctional output of the gut microbiome for therapeutic applications.

Higher consumption of fruit, vegetables, fibre and red wine has beenlinked to higher abundances of beneficial bacteria, includingbutyrate-producing Faecalibacterium prausnitzii. In certain embodiments,desired bacteria, such as SCFA-producers, can also be promoted bymetformin. In certain embodiments, the so-called cross feeding ofbacteria is encouraged to achieve desired butyrate production. Forexample, excess acetate produced by certain bacteria is subsequentlyutilized by butyrate-producing bacteria, such as Faecalibacteriumprausnitzii, Roseburia, and Eubacterium, to produce butyrate. This‘cross-feeding’ effect between Bifidobacterium and butyrate-producingbacteria ultimately leads to an increased butyrate production andaugments beneficial effects, such as improvement of the gut barrierintegrity and pathogen inhibition. The health-promoting attributes ofbutyrate-producing bacteria are supported in numerous diseasedconditions, such as IBD, Crohn's disease, and ulcerative colitis, wherea significant reduction of butyrate-producing bacteria is reported.Though these butyrate-producing bacteria are not directly affected bythe supplementation of oligosaccharides, their butyrate production iselevated due to the increased availability of fermentative end productsgenerated by Bifidobacterium. Thus, in various embodiments, prebioticsplay an important role in mediating complex interactions amongpopulations in the gut microbiota, thus presenting opportunities toachieve therapeutic approaches. Thus, in certain embodiments, a mixtureof bacteria is provided in a probiotic composition to encourage suchcross-feeding effect between Bifidobacterium and butyrate-producingbacteria, such as F. prausntizii. Bifidobacterium utilizes supplementedprebiotics, which stimulates their growth. Acetate produced byBifidobacterium becomes a carbon source for the butyrate-producingmicrobes, stimulating their growth and butyrate-producing activitiesand, in turn, modulating the microbiome function and improving guthealth. Similarly, provision of dietary fiber that can be metabolized bycolonic bacteria into butyrate, achieves the objective of enhancingproduction of this desired short-chain fatty acid (SCFA), which alsoacts as a histone deacetylase (HDAC) inhibitor that epigeneticallyupregulates tumor-suppressor genes in CRC cells and anti-inflammatorygenes in immune cells.

F. prausntizii is not detectable in the fecal samples of babies under 6months of age (Hopkins et al., 2005.). After that, the number starts toincrease gradually, and children of 1 to 2 years of age already have asignificant amount of F. prausntizii in their GI tract. As it isbelieved that babies are born essentially sterile, and receive bacteriafrom the environment immediately upon birth, vaginally delivered infantsreceive their first bacteria as they pass through the birth canal andthus have microbial communities resembling those found in the vaginalmicrobiota of their mothers, dominated by Lactobacillus, Prevotella orSneathia spp. Babies born by Caesarean section (C-section) don't receivevaginal microbes but instead get their first bacteria usually from theskin microbiota of their mother, dominated by taxa such asStaphylococcus, Corynebacterium and Propionibacterium spp. There isincreasing evidence that the early colonization of bacteria affects thehealth of the infants and also influences the host health later in life.It is therefore one aspect of the present invention to provide tobabies, especially those not born vaginally, with a bacterialcomposition that mimics what the baby would naturally experience if itwere born vaginally.

F. prausntizii is one of the most abundant bacteria in a healthy humangut and is believed to have a positive effect on the human gut health.F. prausntizii belongs to the Clostridium leptum group (Clostridiumcluster IV), belonging to phylum Firmicutes (Lineage: Bacteria;Firmicutes; Clostridia; Clostridiales; Ruminococcaceae;Faecalibacterium; Faecalibacterium prausnitzii). F. prausntizii has beenpreviously called Fusobacterium prausnitzii (also cited as F.prausntizii), with it only distantly being related to Fusobacteria andmore closely related to members of Clostridium cluster IV.

Moderate butyrate levels can prevent high-fat-diet-induced insulininsensitivity through epigenetic regulation, and mitochondrialbeta-oxidation. F. prausntizii is one of the unique organisms thatreduce various autoimmune diseases, especially type-1 diabetes via themodulation of gut epithelium homeostasis and immune system. Studiesassociated with gut microbiota and type-1 diabetes have a lowerproportion of butyrate-producing organisms, such as Firmicutes andClostridium, which protects against autoimmune diabetes. While not boundby theory, F. prausntizii is believed to regulate the development ofautoimmune diabetes via butyrate dependent complementary pathways. Anabundant quantity of butyrate can lower the gut barrier function andenhance cell apoptosis, with high levels of butyrate stimulating GLP-1secretion and enhancing insulin sensitivity through cAMP signals, suchas PKA and Epac, which inhibit gastric emptying. Due to the inhibitionof gastric emptying, butyrate can be excreted slowly and accumulates,influencing the anti-inflammatory potential, pH, and oxidative stress.

Butyrate is the major product of carbohydrate fermentation in the colon.Butyrate modulates several processes and is a known anti-proliferativeagent. In cultured cell lines, butyrate inhibits DNA synthesis and cellgrowth, mainly by inhibiting histone deacetylase. Butyrate is alsosuggested to regulate the citric acid cycle, fatty acid oxidation,electron transport and TNF-α signaling. Animal studies have indicatedthat butyric acid may have antineoplastic properties, which means thatit may protect against colon cancer. As dietary fiber is protectiveagainst colon cancer because carbohydrates entering the large bowelstimulate the production of butyrate. Butyrate has also been suggestedto provide protection against ulcerative. F. prausntizii is an importantproducer of butyrate, and the decrease of F. prausntizii has beencorrelated to lower concentrations of fecal butyrate in healthy humansubjects and it is believed that F. prausntizii plays an important rolein the protection of the colon. While not bound by theory, the benefitsof butyrate are thought to depend on several aspects, such as time ofexposure and butyrate amount. Increased butyrate production by F.prausntizii is therefore a desired outcome and employment of CRISPRsystems to achieve the same, employing the known gens involved inbutyrate by F. prausntizii is one important embodiment of the presentinvention.

Studies have shown that there was a statistically significant reductionin the F. prausntizii abundance during both fiber-free andfiber-supplemented diets, but it is postulated that the reduction duringthe fiber-supplemented diet was due to the use of pea fiber, which isnot believed to support the growth of F. prausntizii, and thus, with theproper fiber being employed, the increase in butyrate production isachieved. In situations where there is insufficient fiber for thebeneficial bacteria to consume, the bacteria end up eroding the mucus ofthe gut and leads to epithelial access by mucosal pathogens.

The relative abundance of Bacteroidetes and Firmicutes has been linkedto obesity, with the Firmicutes ratio being significantly higher inobese individuals. It is believed that a high number of F. prausntiziileads to higher energy intake, because F. prausntizii is responsible fora significant proportion of fermentation of unabsorbed carbohydrates inthe gut.

F. prausntizii cultivation has proven difficult because the bacterium isa strictly obligatory anaerobe that does not tolerate any oxygen. Asdescribed herein, encapsulation of F. prausntizii is achieved such thatit can be effectively delivered such that the encapsulated structure candegrade or be fractured at an appropriate time and place to release suchbacteria to a human to derive beneficial results, e.g. the increasedproduction of butyrate. For example, microencapsulation, in a xanthanand gellan gum matrix, and a subsequent freeze-drying protocol can beemployed to achieve this result.

Proton pump inhibitors (PPIs) are among the top 10 most widely useddrugs in the world. PPI use has been associated with an increased riskof enteric infections, most notably Clostridium difficile. The gutmicrobiome plays an important role in enteric infections, by resistingor promoting colonization by pathogens. The differences between PPIusers and non-users are consistently associated with changes towards aless healthy gut microbiome. These differences are in line with knownchanges that predispose to C. difficile infections and can potentiallyexplain the increased risk of enteric infections in PPI users. On apopulation level, the effects of PPI are more prominent than the effectsof antibiotics or other commonly used drugs. PPIs change the gutmicrobiome through their direct effect on stomach acid. This acidityforms one of the main defenses against the bacterial influx thataccompanies ingesting food and oral mucus. PPIs reduce the acidity ofthe stomach, allowing more bacteria to survive this barrier. Species inthe oral microbiome are more abundant in the gut microbiome of PPIusers. Gastric bypass surgery compromises the stomach acid barrier andleads to gut microbiome changes similar to the PP I-associate.

Antibiotics can lead to severe changes in the gut microbiota.Antibiotics are also commonly used in treatment of IBD, even thoughlittle is known about the effects of antibiotics on gut microbiota. Thefecal number of F. prausntizii is lowered in long treatment periods withantibiotics but it is not presently known how antibiotic resistance ofF. prausntizii may affect human health. It is believed, however, that F.prausntizii has a notable impact on gut homeostasis and thus, thesusceptibility of F. prausntizii to different antibiotics is believed tobe important in the treatment of various ailments. Provision ofadditional F. prausntizii after a regimen of antibiotics is thereforeone aspect of various methods of the present invention.Antibiotic-induced changes in the gut microbiota are usually temporary,but long-term microbial population fluctuations have also been reported.It is believed that antibiotics may even move the gut microbiota to anew, alternative stable state. Antibiotic-induced alterations in the gutmicrobiota raise the disease risk by increasing the susceptibility topathogen colonization; for example, diarrhea caused by Clostridiumdifficile is a well-known consequence of antibiotic courses. The use oflive F. prausntizii is preferred due to the greater immuno-stimulatoryeffects of live F. prausntizii, via TLR2 activation. It is believed thatthis effect is potentially linked to its barrier maintaining properties.It is butyrate, instead of other substances produced by F. prausntizii,that exerts significant anti-inflammatory effects observed, and it isbelieved that the target of butyrate is histone deacetylase 1 (HDAC1).

In other embodiments, the bacterial composition employed includes bothF. prausntizii and Akkermansia muciniphila, another abundant member ofthe human gut microbiota. It is further believed that Faecalibacteriumprausntizii plays a vital role in diabetes and can be used as anintervention strategy to treat dysbiosis of the gut's microbialcommunity that is linked to the inflammation, which precedes autoimmunedisease and diabetes.

The microbiota in adults is relatively stable until the persons get 60years old. Gut alterations lead to elevated gut permeability and reducedgut mucosal immunity, contributing to the development of variouscancers, autoimmune disorders, inflammatory bowel diseases, metabolicsyndrome and neurodegenerative diseases. The resultant elevatedintestinal permeability is a consequence of reduced expression of tightjunction proteins that favors the uncontrolled passage of antigens andenables the translocation of bacterial lipopolysaccharide to the gutconnective tissues and to the blood circulation, causing insulinresistance and metabolic endotoxemia.

The gastrointestinal tract pH normally ranges between 5 and 5.5 in theileum and the colon has a range from 6.6 to 7.0, which is one of themain factors in constructing the shape of the microbial communities inthe colon. Diet compositions containing fermentable polysaccharides areregulators of the intestinal pH, which facilitates a more acidicenvironment through the end-products of SCFAs in the gut.

Stool pH becomes more alkaline with the increase in age and differssignificantly between genders with higher consumption of animal proteinbeing one possible mechanism for higher pH. Such alkalinity is generallycaused due to its alkaline metabolites produced by proteolyticputrefactive bacteria, such as Bacteroides, Propionibacterium,Streptococcus, Clostridium, Bacillus, and Staphylococcus.

An individual generally represents a unique collection of genera andsub-species and it may be different based on the diet (vegetarian orWestern with high protein or fat), the age of the host organism, geneticand environmental factors. Diet greatly influences the diversity of themicrobiota in the gut and the microbiota is genetically well equipped toutilize various nutritional substrates to maintain a normal gutmicrobiota pattern. An adequate SCFA (butyrate) production level isessential for gut integrity and butyrate-producing bacteria, such asEubacterium, Fusobacterium, Anaerostipes, Roseburia, Subdoligranulum,and Faecalibacterium, but especially, F. prausntizii, have the potentialof anti-inflammatory effect and help to reduce bacterial translocation,improve the organization of tight junctions and stimulate the secretionof mucin to maintain the integrity of the gut, with beneficial effectsagainst inflammation in the gut.

Inflammation is one of the major pathophysiological factors leading toinsulin resistance and progressively causes type-2 diabetes. F.prausntizii counts significantly decreased in diabetic individuals withnegative correlation to glycated hemoglobin HbA1c values. Along withAkkermansia muciniphila, F. prausntizii is abundantly found inindividuals with normal glucose tolerance compared to the pre-diabeticsubjects. F. prausntizii can convert acetate into butyrate usingbutyryl-CoA: Acetate CoA-transferase (BUT) pathways, thereby providing abalanced pH in the gut.

While specific embodiments and applications of the present inventionhave been described, it is to be understood that the invention is notlimited to the precise configuration and components disclosed herein.Various modifications, changes, and variations which will be apparent tothose skilled in the art may be made in the arrangement, operation, anddetails of the methods and systems of the present invention disclosedherein without departing from the spirit and scope of the invention.Those skilled in the art will appreciate that the conception upon whichthis disclosure is based, may readily be utilized as a basis fordesigning of other methods and systems for carrying out the severalpurposes of the present invention to instruct and encourage theprevention and treatment of various human diseases. It is important,therefore, that the claims be regarded as including any such equivalentconstruction insofar as they do not depart from the spirit and scope ofthe present invention.

What is claimed is:
 1. A method for reducing the likelihood ofdeveloping liver cancer in an individual diagnosed with non-alcoholicfatty liver disease, comprising: providing in the gut of an individual apopulation of beneficial bacteria selected from the group consisting ofLactobacillus species; administering at least 6 grams per day of fiberto the individual to maintain a therapeutically effective amount of thebeneficial bacteria in the gut of the individual; increasing the levelsof Roseburia, while reducing the levels of Akkermansia spp. in theindividual's gut microbiome, and administering an effective amount of acomposition comprising modified Lactobacillus reuteri bacteria, whereinthe Lactobacillus reuteri bacteria is modified using CRISPR-Cas and/orCpf1 systems, to provide said modified Lactobacillus reuteri bacteriawith the ability to survive conditions in the duodenum or jejunum of theindividual's small intestine.
 2. The method as set forth in claim 1,further comprising inhibiting expression ofdiacylglycerolacyltransferase-2 (DGAT-2) in said individual.
 3. Themethod as set forth in claim 1, wherein the beneficial bacteria arepresent on a thin film mucosal strip.
 4. The method as set forth inclaim 1, wherein the population of beneficial bacteria include bacteriathat have been modified to increase the level of butyrate.
 5. A methodfor reducing the likelihood of developing liver cancer in an individualdiagnosed with non-alcoholic fatty liver disease, comprising: providingin the gut of an individual a population of beneficial bacteria selectedfrom the group consisting of Lactobacillus species; administering fiberto the individual to maintain a therapeutically effective amount of thebeneficial bacteria in the gut of the individual; and administering atherapeutically effective amount of a bacterial formulation comprisingFaecalibacterium prausnitzii and at least one of Coprococcus,Veillonella, Roseburia, Bifidobacterium, and Prevotella.
 6. The methodof claim 5, further comprising; providing inulin in an amount sufficientto reduce the pH in the colon of the individual and acidifying of thecolon to enhance intestinal Ma.sup.2+ absorption by the individual. 7.The method of claim 5, wherein the Faecalibacterium prausnitzii bacteriaemployed are from the individual treated and are first isolated from theindividual's stool.
 8. The method of claim 5, further comprising,employing a clustered regularly interspaced short palindromic repeats(CRISPR) CRISPR associated protein (Cas) system CRISPR/Cas to ridPrevotella bacteria of virulence factors selected from the group offimbria, hemolysins, adhesions and hemagglutinins and administration ofsaid Prevotella bacteria to the individual.
 9. The method of claim 5,further comprising, administering a therapeutically effective amount ofbacteria of the Bacteroides family that have been modified to reduce theamount of a ligand-activated transcription factor.
 10. The method ofclaim 5, further comprising, increasing the proportion ofRuminococcaceae in the individual's gut microbiome.
 11. The method ofclaim 5, further comprising, reducing the proportion of Escherichia inthe individual's gut microbiome.
 12. The method of claim 5, furthercomprising increasing the levels of Leuconostoc, Lactococcus, and/orPediococcus.
 13. The method of claim 5, further comprising,administering an effective amount of a composition comprising modifiedLactobacillus reuteri bacteria, wherein the Lactobacillus reuteribacteria is modified using CRISPR-Cas and/or Cpf1 systems, to providesaid modified Lactobacillus reuteri bacteria with the ability to surviveconditions in the duodenum or jejunum of the individual's smallintestine.
 14. The method of claim 5, further comprising, modifyingbacteria of the Bacteroides family to produce reduced amounts of aligand-activated transcription factor as compared to non-modifiedbacteria.
 15. A method for reducing the likelihood of developing livercancer in an individual diagnosed with non-alcoholic fatty liverdisease, comprising: providing in the gut of an individual a populationof beneficial bacteria selected from the group consisting ofLactobacillus species; administering fiber to the individual to maintaina therapeutically effective amount of the beneficial bacteria in the gutof the individual; and administering a therapeutically effective amountof a bacterial formulation comprising Faecalibacterium prausnitzii, andadministering an effective amount of a composition comprising modifiedLactobacillus reuteri bacteria having the ability to survive conditionsin the duodenum or jejunum of the individual's small intestine.
 16. Themethod of claim 15, further comprising, acidifying of the colon of theindividual to enhance intestinal Ma.sup.2+ absorption by the individual.17. The method as set forth in claim 15, further comprising inhibitingexpression of diacylglycerolacyltransferase-2 (DGAT-2) in saidindividual.
 18. The method as set forth in claim 15, wherein saidbeneficial bacteria are encapsulated in a frangible enclosure.
 19. Themethod as set forth in claim 15, further comprising increasing thelevels of at least one of Roseburia, Coprococcus, Veillonella,Bifidobacterium, and Prevotella in the individual's gut microbiome. 20.The method as set forth in claim 15, further comprising, reducing thelevels of Akkermansia spp. in the individual's gut microbiome.